KR100420377B1 - New Uses of Lysozyme Dimers - Google Patents

Abstract

The present invention relates to pharmaceutical compositions comprising lysozyme dimers of high purity, eg, up to 10 wt% of unwanted products, new uses thereof. Such uses include topical, oral and parenteral administration of compositions for general immunostimulation such as the prevention and treatment of diseases of humans and animals, including cancer, hair growth diseases, fish diseases and bee disease. A single or repeated administration of lysozyme dimer composition, preferably at a concentration of 5-500 μg / kg, induces general stimulation of the immune system.

Description

New Uses of Lysozyme Dimers

In the past 80's, it was found that certain enzymes are in dimeric form, less toxic than monomers, while retaining the beneficial properties of the same monomers and in some cases have no adverse effects when used as therapeutic doses.

Antiviral and antibacterial compositions consisting of the active ingredient lysozyme dimer or other dimeric enzymes are described in WO89 / 11294. Lysozyme dimers have been reported to inhibit the growth of many bacterial strains cultured in samples taken from patients when used at concentrations of 1.25-20 mg / ml culture. In addition, dimers have been reported to be effective in treating canine parvovirus infection when administered orally at 1-2 mg / kg twice a day. A further feature of lysozyme dimers was later discovered, e.g. the therapeutic use of additional drugs in particular for the treatment of bacterial and viral infections, as detailed in WO94 / 01127.

WO94 / 01127 proposed a model theory that may be helpful in understanding the various effects observed in lysozyme dimers. The overall mode of action of lysozyme is not yet fully understood but there is additional therapeutic activity that cannot be explained by the lytic activity of the corresponding monomer. The inventors have discovered the specific immune stimulatory effect of dimeric lysozyme, particularly dimer lysozyme, which is associated with regulating levels of cytokine levels. In addition, from their experiments, it was concluded that lysozyme dimers inhibit the invasion of bacterial cells by viruses by blocking certain parts of the outer cell surface that appear to be composed of virus soluble proteins.

The existing literature details the results obtained in lysozyme dimers in vitro. In particular Bartholeyns and Zenebergh (Europ. J. Cancer, Vol. 15, 1979, 85-91) tested dimer lysozyme for cytostatic activity against liver cancer cells in vitro. In addition, it was observed that cancer cell proliferation was inhibited by 73% ± 15% in cell culture (ibid., P. 89, Table 2).

Surprisingly, no reports have been reported for in vivo experiments on lysozyme dimers with the exception of WO94 / 01127. Surprisingly, Bartholeyns and Zenebergh or other researchers did not promote the discovery that they could fight cancer. Sorrentino et al., Eur. At least one possible reason in an experiment (FIG. 1) comparing the purity of lysozyme dimers produced according to the method of J. Biochem, 124, 183-189 (1982) with the purity of lysozyme dimers suitably used in the present invention (FIG. 1) can see;

While by-products such as lysozyme monomers, trimers, tetramers are found in high concentrations in formulations made according to Sorrentino, the products in the present invention are very pure, i.e. up to 90% of the total lysozyme portion of the formulation. It contains a sieve. The process for producing such high purity lysozyme dimers is described in detail in WO 91/10731.

This confirms that the purity of existing technology lysozyme dimers is not suitable for in vivo experiments and applications, despite the beneficial antibacterial activity of lysozyme 15 years ago, lysozyme monomers are rather toxic, causing inflammation, severe allergies and toxic shock symptoms. Can be.

In this situation, other researchers, including Bartholeyns and Zenebergh, have said that additional studies of lysozyme dimers should be conducted to develop lysozyme dimers that can be used in vivo. I can better understand why.

Despite the lack of scientific research activities due to the bias of existing techniques for the use of lysozyme dimers in vivo, the present inventors have improved the production and purification of lysozyme dimers and the antiviral and antibacterial properties detailed in WO94 / 01127. The research and development work was carried out to find a method that can be applied to humans and animals in vivo as a product that can be applied.

Based on the low toxicity of dimer lysozyme compared to monomers and the availability of novel high-purity lysozyme dimer preparations, the inventors have found that lysozyme dimer preparations in vivo, although existing techniques do not suggest their use in treating diseases other than viral or bacterial infections. Chemotherapy was attempted. One of the greatest advantages of the present invention is its use in replacing the highly toxic anticancer agents used in conventional chemotherapy with high purity lysozyme made from pharmaceutical compositions.

The present invention relates to novel uses of lysozyme dimers and compositions comprising such dimers. New applications are useful for the prevention or treatment of symptoms or diseases in which the natural defense and regeneration system is impaired, especially in humans and animals, based on the general stimulation principle of the immune system.

Figure 1 compares the purity of lysozyme dimers made using two different model techniques.

High purity lysozyme dimers can be used in the manufacture of pharmaceutical plastics that can be used directly for the treatment of humans and animals because of their lower toxicity than monomers.

As described above, such high-purity lysozyme dimers contain less than 10% wt of by-products and can be obtained through dimerization of monomers derived from humans, animals, eggs, plants, microorganisms, and the like. Monomers are isolated in their natural form or are prepared by chemical or genetic engineering methods to separate monomers having the same biological or chemical properties as naturally occurring lysozyme.

In in vitro cell culture and in vivo test models, suitably in rat test models, the inventors have found that the lysozyme dimer compositions employed have a strong ability to inhibit the cell proliferation of cancer tissues in vitro and in vivo. Could be explained as. Such therapeutic actions could be identified in a variety of known cancer types affected by or affected by viruses. These viruses include, in particular, human papilloma virus, Ipstein-Barr virus, hepatitas B virus, HTLV- (adult T-cell leukemia virus), including subtypes, HTLV-II (lymphoma virus) and HIV- ( Retroviruses such as human immunodeficiency virus).

The forms of cancer induced or affected by the action of these viruses consist of cervical sarcoma, urinary sarcoma, Kaposi's sarcoma, major hepatocellular sarcoma, leukemia, T-cell lymphoma, AIDS or subclass thereof. It is an object of the present invention to provide pharmaceutical compositions and methods for the prevention and treatment of cancer in humans and animals.

Another beneficial application of the high purity lysozyme dimer is when the lysozyme dimer composition is administered to a patient with post-operative wounds, such as lower amputation surgery. It can be seen that topical administration of dimers successfully treats wound infections and promotes and regenerates hair growth in the treated areas.

This surprising finding led to further investigation and finally produced pharmaceutical compositions that can be used for hair growth disorders, especially hair growth disorders based on immunological dysfunction or dysfunction, such as alopecia areata.

Such a composition can perform to some extent a long-awaited composition that can be used as a hair growth stimulant. The basic principle of this unexpected effect appears to be due to the stimulation and improvement of immune defense mechanisms, the improvement of blood circulation in epithelial tissue and skin, the general improvement of immunological function and some unidentified effects.

Another surprising and commercially interesting part of pharmaceutical compositions comprising lysozyme dimers can be found in fish farms and apiaries where they can be replaced by different non-specific immunostimulants including lysozyme dimers in conventional antibiotic experiments. Can be.

The use of immune stimulants in fish culture for the prevention and treatment of fish disease ensures new developments (Siwicki and Anderson 1990; Siwicki et al. 1994). Currently, substances for treating fish diseases include antibiotics, drugs and chemicals used to sterilize fish tanks (Stoskopf, 1993). Such treatment may be at least partially effective in the treatment of certain diseases, but the problem arises of the emergence of pathogenic microbial strains resistant to the accumulation of such substances in the environment and to fish and various antibiotics.

In the case of beekeeping, killing bees is common when infected by microorganisms such as bacterial and / or viral infections. This treatment prevents the spread of infection between different colonies and minimizes losses. Thus, there is a need for another reliable method for the prophylactic and / or therapeutic treatment of honey that combines the benefits of antibiotics to further enhance the immune system of bees and antibiotics. This does not make antibiotic-resistant microbial strains. The present invention provides a method that meets these needs. In addition, even if the dimer lysozyme is in the final honey at a detectable concentration, it can be seen as an improvement in quality rather than worse because of the general immunostimulation of the lysozyme dimer.

In general, immunostimulants are composed of biological and synthetic compounds that can enhance unspecified cells and human defense mechanisms in humans and animals. Beta-glucans, chitosan, levamisol, residual minerals and mineral complexes, and various substances derived from many plants and animals are effective in preventing disease. Some forms of betaglucan are specific for stimulating unspecific immune responses in the body fluids of fish cells.

Unspecified defense mechanisms, including phagocytosis and the production of oxidative radicals, are rapidly activated by immunostimulatory substances and rapidly to protect fish against pathogenic microorganisms such as viruses, bacteria, mycoplasm and fungi against parasites or other pathogens. Ready. Thus, this action takes precedence over certain immune responses that require a significant period of time to produce a specific adopted immune response, including antibody composition and specific cell activation.

Results obtained from primary in vivo trials showing significant improvement in immunological defense mechanisms are detailed investigations of the effects of lysozyme dimers on fish-specific, trout, salmon-specific and specific cellular and humoral defense mechanisms and bee defense mechanisms. The program was launched.

Accordingly, it relates to novel uses of lysozyme dimers that can be used in the manufacture of pharmaceutical compositions for stimulating unspecified and humoral immune responses in fish and honey.

Another object of the invention is a pharmaceutical comprising a lysozyme dimer capable of inducing unspecified stimulation of the immune system by single or repeated use of the composition for the prevention and treatment of diseases that can occur naturally in fish and bees. Provided are methods for using the composition.

In biological experiments, when damaged by certain compounds or other bioactive substances, such as by-products of pharmaceuticals administered to fish, the fish is free of lysozyme monomers and contains high purity lysozyme dimers containing less than 10% wt of unwanted by-products. WO 91/10731) seems to be appropriate. In order to reduce breeding costs in fish farms or apiaries, it is also possible to administer lysozyme dimers of the lowest possible purity, which are applied in dosages that do not cause side effects due to the toxicity of by-products, in particular lysozyme monomers.

Lysozyme dimers referred to herein can be administered directly to a patient in need thereof or used in the manufacture of pharmaceutical compositions, usually in the form of gallens. Gels, ointments or liquid compositions may consist of lysozyme dimers at a concentration of 0.01-10 mg / ml and may sometimes contain 0.1-1.0 mg / ml. These are sterile; It may be made of a pyrogenic composition and optionally include at least one physiologically acceptable solvent and / or receptor and / or at least one suitable preservative.

Pharmaceutical compositions comprising lysozyme dimers are intended for topical or parenteral use consisting of external use to the body surface including topical injection or subcutaneous injection around a solid tumor. Intravenous injection may be replaced in the therapeutic course or by additional local topical treatment.

However, it is understood that the lysozyme dimer composition is sufficiently administered to the mucosa through inhalation of the liquid composition (nasal, oral, pharyngeal mucosa) or topical use of the liquid or cream composition (vaginal, cervical mucosa) or tampons containing lysozyme dimer material. Proved.

In some cases, lysozyme dimers are administered orally in conventional herbal form, such as instant tablets, capsules or dragees, or provided in pellet, particle, phlox or powder form. Such solid compositions often comprise 0.01-10 mg active drug, suitably 0.01-1.0 mg per g of total composition. It may also suitably further comprise at least one additive or conventional additives such as perfumes or colorants. In the case of treating fish, the solid composition may further include nutrients. Or in addition, the lysozyme dimer can be dissolved in the fish tank, and the active drug can be reabsorbed through the fish gills. In the case of beekeeping, the lysozyme dimer composition can be used by dissolving the solid composition in water, tea, sugar solution or other conventional liquid which is prepared as a feed substitute for making honey. However, it is advisable to mix the honey with an appropriately concentrated-aqueous solution to allow the honey to take the drug.

However, oral compositions can also be used in osmotic systems. In certain cases, topical use of sterile dressings or tampons containing high purity lysozyme dimer effective amounts to prevent or treat cancer of the urethra can be beneficial.

Various forms of the aforementioned lysozyme dimer compositions are administered in single or repeated doses at 0.005 to 0.5 mg / kg body weight, particularly in doses of 0.01 to 0.1 mg / kg. The required concentration of active lysozyme dimers in the final pharmaceutical composition should, of course, be tailored to the type of treatment or prophylaxis tailored to the patient concerned and to the size of the patient or animal. In most cases, however, the aforementioned concentration ranges are sufficient for proper treatment. Nevertheless, in veterinary medicine, the concentration of active lysozyme dimers in the composition may be increased to 10 mg / ml or 20 mg solution ml or ointment above and below the solubility of the dimer in the relevant solvent.

Where possible, the combined course of administration of the compositions described above is suitably oral, rectal or topical.

Because of the essential non-toxicity of high purity lysozyme dimers, compositions containing such dimers can be used for many months or years without dangerous side effects. The prophylactic or therapeutic intervals of drug range once or twice daily or in the morning or monthly dosage range and may be longer depending on the condition and emergency treatment of each patient as well as the immunostimulatory effect by lysozyme dimers.

As mentioned above, the present invention is at least partly possible using higher, lysozyme dimers. Surprising differences due to the quality of the product, in particular the sharing of undesirable lysozyme monomers, are illustrated in FIG. 1; Line 1 represents the LMW prestained protein standard. Phosphorylase B142,000 Dalton, BSA 97,000, ovalbumin 50,000, carbon anhydrase 35,100, soybean trypsin inhibitor 29,700, lysozyme 21,900 (Biorad, USA); Lines 2 and 3. The purified lysozyme dimer ^{LYOIUM KLP ⓡ 602 (KLP-602} ), Lot 506449, laboratory control; Line 2 is 6.6 μg, line 3 is 19.8 μg; Lines 4 and 5 show another KLP-602 batch: 6.6 μg in line 4, 19.8 μg in line 5; In lines 6 and 7, Sorrentino et al., Eur. J. Bioch. 124, 183-189 (1982) shows a lysozyme dimer preparation (KIW-607), which is 6.6 μg in line 6 and 19.8 μg in line 7.

Pure lysozyme dimer formulation KLP-602 contains four times more dimer than the comparable product KIW-607, while comparable product KIW-607 contains six times more monomers than KLP-602.

In order that the present invention described above may be fully understood herein, the following examples are presented. Such embodiments are for illustrative purposes only and are not intended to limit the invention thereto.

Example 1

Lysozyme dimer (KLP-602) for the treatment of lymphoid leukemia in AKR rats.

Materials and methods

In this study, a 7-month old AKR (male and female) Ruchymia strain inoculated with Graffi virus obtained from Inbreeding Animal Center, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, and Wroclaw was used. In AKR rats, endogenous leukemia virus is initially activated, which becomes clinically evident at 7-9 months. Mice are divided into three groups of 30 animals each;

1 group (experimental group)

Animals are administered subcutaneously with KLP-602 once at a dose of 20 μg / kg 0.3 cm ³ PBS.

2 groups (experimental group)

Animals are administered KLP-602 twice in the same amount as in group 1 above.

3 groups (control)

Animals are dosed twice with 0.3 cm ³ PBS.

Mice are killed by bleeding for 3 to 6 weeks after the last KLP-602 injection (10 mice in each group). Ten mice in each group survive.

Perform the following inspection.

1. Determine the ratio of organ and total body weight by measuring total body weight and the amount of internal organs and external cervical lymph nodes such as liver, spleen, and thymus. Therapeutic activity ratio is assessed using L / K × 100%, where L means survival time of KLP-602 treated week and K means average survival time of control.

2. A biopsy of the liver, spleen, thymus, and lymphocytes is done for histopathology. The part is colored with H + E of reticulated fiber mori method.

3. The level of free radicals in serum is assessed using the chemiluminescence (Chl) method, which can detect very weak photon emission in serum.

4. Multinuclear phagocytosis (PMN) is assessed using NBT and chemiluminescence (Chl) tests.

result:

Subcutaneous administration of leukemia AKR rat strains (group 1) and twice (group 2) at a dose of 20 μg / kg significantly increased body weight when compared to the control group (group 3) measured 3 weeks after the study. The amount of internal organs such as spleen, thymus, and lymphocytes was significantly increased.

Six weeks after the study, the amount of spleen, thymus and lymphocytes in group 1 decreased and liver and thymus increased in group 2 when compared to controls (Table 1).

Table 2 shows the ratio of the internal organ weight to the total body weight. In comparison with the two groups (groups 1 and 2), an increase in the amount of thymus after 3 weeks was seen in group 3 (control). Six weeks later, the amount of spleen, thymus and lymphocytes increased in groups 2 and 3 compared to group 1.

Pathological histology after 3 and 6 weeks revealed mild colonization (experimental) and excessive colonization (control) of cervical lymphocytes, thymus and spleen. However, leukemia infiltration in the liver was found only in the control group. Lymphocytes have an elliptical or circular polysomy nucleus with a number of heterogeneous similar features. These cells are accompanied by weakly differentiated central blasts, reticulum and proliferating megakaryocytes in the spleen. Lymphatic organs, such as spleen, lymphocytes and thymus, contain weak thin reticulated fibers.

Chemifluorescence;

Three weeks after the study, free radical activity in the serum of control rats was K = 18 × 10 ⁻³ . The activity of free radicals in group 2 was low (K = 10 × 10 ⁻³ ). After 6 weeks the free radicals were K = 10 × 10 ⁻³ in control animals, lower than group 2 (K = 16 × 10 ⁻³ ) and slightly higher than group 1 (K = 1.5 × 10 ⁻³ ). Phagocytosis activity of PMN can be seen to be significantly increased in all experimental groups after 3 weeks compared to the control group; 4688 Chl (Group 1), 3485 Chl (Group 2). 2052 Chl. After 6 weeks, phagocytic activity was equally high in group 2 (3264 Chl) and lower in group 1 (2477 Chl). There was a significant increase in phagocytic activity measured by the NBT test after 3 weeks in the treatment group and 0.27 and 0.26 in each group 1 and 2. After 6 weeks, phagocytic activity was low and only in group 2. Phagocytic activity is 0.22 in the control group. The KLP-602 therapeutic efficacy indices in groups 1 and 2 were at the lowest levels of 128% and 150%, respectively. This indicates the prominent immunostimulatory properties of KLP-602 in further studies.

Leukemia infiltration is significantly smaller in the experimental group than in the control group, but absent in some rats. Leukemia infiltration is in the thymus, spleen and lymphocytes. There is also liver of control animals. Studies show that KLP-602 can inhibit or delay leukemia in AKR mice. In contrast, rapid growth of lymphoid organs occurred in animals treated with placebo with morphological changes that were differentiated lymphocytic leukemias.

KLP-602 also inhibits the growth of free radicals in rat plasma. It inhibits free radical processes similar to homeostatic activity such as vitamin E, C, or selenium. Inhibition of leukemia cell proliferation in AKR mice treated with KLP-602 may depend on inhibition of free radical induced processes. In control rats (without KLP-602), free radical levels are higher and appear to be related to the extent of the disease.

KLP-602 also stimulates the phagocytic activity of the mononuclear phagocytosis system (MPS) measured by two independent methods, NBT and Chl, to stimulate cell-mediated immunity. It also regulates TNF synthesis. In addition, “tumor rejection antigen” on the surface of leukocyte cells can be recognized by T-cells associated with MPS. Among these T cells are tumor anti-invasive lymphocytes (TILs) that are 50-100% more effective than lymphocyte-activated killer (LNK). They have a CD ₄ or CD ₈ or mixed phenotype and they express the IL-2 receptor on the surface. It is not yet known why they are paralyzed by neoplastic cells.

MPS cell stimulation and the reticulum-endothelial system are essential immunological and antiviral mechanisms. Such stimulation of cells produces inteferol and indirectly activates specific or nonspecific immunological processes, B-cells, T-cells or NK dependence.

MPS cells participate in immunological processes through Fc receptors, C ₃ receptors or HLA receptor-II and lectins, transpecrine, neurokinase, insulin and 50 other lesser known receptors. Any change in their expression and affinity indicates cell activity. MPS cells can phagocytosis NO-sensitive cells because they synthesize such substrates. Activated phagocytes are called "inflammatory phagocytosis" or "blood dry" cells. MPS cells can destroy viruses through their cytotoxic and cytolytic, protective virion production and interferol synthesis.

Preliminary studies in AKR rats indicate that KLP-602 can be successfully used to treat leukemia in AKR rats.

Example 2

Effect of High Purity Lysozyme Dimer Formulation (KLP-602) on Hair Growth in Alopecia areata

This study was carried out to determine whether the inclusion containing KLP-602 is active against hair growth in alopecia areata.

Materials and methods;

This study is a controlled comparative clinical study.

The study generally involves 12 patients (two males and ten females) with "terminal" alopecia in good health. The age distribution is from 19 to 50 years old. The MONOXIDIL 12 patients (3 men and 5 women patients) with alopecia areata treated with the ^ⓡ to the control group. These ages range from 19 to 49 years. The population distribution, such as age and gender, in the two treatment groups was similar in the two groups.

Patient selection criteria

Inclusion Criteria

1. Alopecia areata at the "end stage" stage

2. 18-50 years old

Exclusion Criteria

1. Alopecia areata of "dystrophy"

2. Different forms of alopecia areata

3. other skin diseases

Concomitant drug;

Drugs are not allowed unless required to treat a general state of health.

Process number assignment;

During the study, the treatment groups are numbered sequentially in the treatment group. Enter the appropriate number on each case report form (CRF) at the appropriate location.

Drug

According to the inherent shape, the hair fragrances prepared by NIKA are placed in the same plastic bottle with a small graduated tip dropper. Follow your dermatologist's instructions to apply 0.05% KLP-602 lotion to your headband three times a day. Treatment begins immediately after the dermatologist recognizes that alopecia areata.

2% MINOXIDIL ^ⓡ solution was rubbed twice a day to affected areas.

The treatment period is 16 weeks. The patient is observed for the next two months after treatment.

Stability evaluation.

Test Population Screened by overall medical history and medical examination at the beginning and end of the study in all patients. Blood tests and urine tests are done by routine laboratory tests before and after treatment. Laboratory studies include:

Hematology:

Hemoglobin, hematocrit, total and differentiated white blood cell count

Serum biochemistry.

ALT (GPT), AST (GOT), alkaline phosphatase,

Bilirubin

Urine;

Dipstick urine tests for protein, blood and glucose.

Patients are observed once a month for four months of treatment and the next two months. At each visit the patient is asked questions about irritation, burns, itching, etc. due to general or local reactions; You will be tested for erythema or other local side effects.

Evaluation of effectiveness.

Evaluation variables

1. Hair growth is the primary effect factor.

2. The healing time of the head leather is the secondary effect factor.

3. Photographic examination at each time next in the study.

-Before using KLP-502 Hair Color.

-Hair growth

-When the use of KLP-602 hair perfume

Microscopic examination (tricogram) is performed. Standard tricograms of hair taken from a wide range of alopecia areata provide the most useful information and measure the severity and therapeutic effects of the disease.

At each visit, signs of hair growth are assessed based on the following criteria.

- none

Slightly-Hair growth (brasion or some scattered hair).

Medium-Incomplete (partial)

- very

"Externally acceptable" hair growth means that it can cover the scalp sufficiently and hide the remaining portion of the hair injury.

result:

Enrolled in the study (12 patients treated with 0.05%, KLP-602, 12 of controls treated with 2% Minoxidil solution)

It was completed during the four month treatment period and for the next two months.

effect.

The most useful measure of effect is the investigator's estimate at the end of the study. Overall, 8 out of 12 (66%) in KLP-602 and 6 out of 12 (50%) in minoxidil showed significant hair growth during the study. Of these, four in the KLP602 (33%) and three in the minoxidil (25%) appear to have completely cosmetically acceptable hair growth. Partially hair growth was seen in the KLP-602 phase (3 out of 2 (25%) and in the minoxidil phase 3 (25%). In at least one patient in the KLP-602 population (9%) There was no hair growth in 4 out of 12 (33%) of KLP-602 population and 6 out of 12 (50%) of minoxidil group.

There was hair growth in all 8 body weights in KLP-602 in the group with alopecia areata with 25% scalp loss, and 6 in 7 in the minoxidil group. There was no regeneration in alopecia areata cases with scalp loss of more than 25% in both treatment groups.

The average time of hair growth correlates with the duration of treatment ranging from 1 month to 3 months in both groups. In the KLP-602 population, these individuals responded one month after treatment. After one month in one female subject, hair growth was 5 mm long at the tip of the hair and the bald part was densely covered with regenerated hair.

At 2 months of the control visit, a female had alopecia (emotional stress-divorce) and no response to further treatment. A negative result was shown. Hair regeneration occurred one month after treatment in a group of minoxidil. Four patients in the KLP-602 group had hair regeneration two months after treatment. Three months after the study, two patients in the KLP-602 group and three in the minoxidil group had hair regeneration.

stability.

There were no common side effects during and after the treatment. There were no abnormalities in laboratory blood and urinalysis at baseline at the end of the study in both treated groups.

There were local side effects during the study: A woman in the KLP-602 population complained of scalp burning and itching at the second month of treatment. She wore a new wig for this condition. The problem disappeared within two weeks without further treatment, and she stopped the treatment and the results were good. One subject with 2% minoxidil solution complained of sticky hair, but there were no significant clinical signs on the scalp test. The patient is involved in this attempt but continued treatment.

No significant correlations were found between the response organs and hair regeneration from one week up to one year, which were short but similar in patient age, baldness, and in both groups. All non-responders in both treatment groups had alopecia areata for at least 5 years (5-15 years).

Argument.

This study evaluated the stability and effect on hair growth by applying 0.05% KLP-602 lotion topically to patients with alopecia areata and compared it with treatment with 2% minoxidil solution for 4 months.

Hair flavors containing KLP-602 have been well adapted to patients. 66% of patients treated with 0.05% KLP-602 lotion increased hair growth, compared to a 50% increase in patients in the minoxidil population.

Cosmetically acceptable hair growth was 33% in the KLP-602 group and 25% in the minoxidil group. For alopecia areata with less than 25% modified scalp, better results were obtained.

The 0.05% KLP-602 lotion is easy to apply topically and does not cause topical or general side effects. Given the short-term study (16 weeks), the results obtained are encouraging.

conclusion.

1. Hair oil containing KLP-602 is very soft and does not cause any side effects.

2. 0.05%, local treatment of KLP-602 is safe and easy.

3. There were no local or general side effects at the laboratory level during the study.

4. Short-term therapy (4 months) and the next two months, although this period may be sufficient to assess the effectiveness of the treatment, it is not possible to fully evaluate the treatment results and implement the treatment.

5. In the results obtained in this trial, the 0.05% KLP 602 lotion is comparable to the 2% minoxidil solution and is quite effective for the treatment of alopecia areata.

6. In the results and clinical investigations obtained from the study, topically treated KLP-602 induced hair growth in patients suffering from alopecia areata.

summary.

This study was conducted to evaluate the effect and endurance of hair balm containing KLP-602 in alopecia areata. Applying 0.05% KLP-602 lotion three times a day generally appears to adapt well during the four month treatment period. There were no local or computational side effects. There was a better hair growth response in the KLP-602 population than in the minoxidil population. Hair growth responses were seen in 66% of patients who received 0.05% KLP-602 lotion. The proportion of patients with moderate or dense hair growth responses is greater in bald patients with scalp loss of less than 25%. It can be seen that 0.05% KLP-602 is an excellent agent for alopecia areata.

Example 3

Prophylactic application of lysozyme dimers to induce general stimulation of the immune system of fish.

Testing method.

Three hundred healthy rainbow trout (Oncorhynchus mykiss, family of salmonidae) weighing 90-100 g each are included. The lysozyme dimer, prepared according to WO91 / 10731 (herein "KLP-602"), contains about 10% unwanted monomer lysozyme and is essentially free of monomeric lysozyme, from 10 to 10 body weight per body to be suitable for intraperitoneal injection into fish. Dissolve in PBS to make an injection to administer 100 μg lysozyme. The drug is administered once a day. Therapeutic course consists of different groups of treatment of the drug on day 1, day 3 or day 1,3,5. Control fish do not receive lysozyme dimers.

One, two, three, four weeks after the last injection, 10 fish are taken from each treatment and control group. The fish are immobilized and 100 ml blood is collected from the dorsal vein using heparinized Greiger. Determine phagocytosis of PMN and MN cells, respiratory rupture and potential lethality of PMN and MN cells, MPO activity in PMN phagocytes and lysozyme and gamma-globulin levels in serum.

In addition, 20 of each group will undergo a disease (chorus) challenge test. In fish, intraperitoneal injections of aeromonas salmonicida bacterial suspensions grown in nutrient media for 48 hours. Animal mortality is determined and pathogens are isolated from the kidneys of dead animals to correlate with applied bacterial infections.

result.

In the KLP-602 treated population, all of the immunological variables described above were significantly increased (p <0.05) compared to the corresponding control. This surprising result was seen in all groups, for example, in all groups observed after 1-, 2-, 3-fold application of KLP-602 and up to 4 weeks after the final injection.

Fish mortality in the KLP-602 treatment group was significantly lower than that in the control group. Respiratory rupture activity is relatively high, which leads to the dietary index and neutrophil myeloperoxidase (MPO) levels of the population treated by two or threefold application of KLP-602.

A. The results observed in the known rainbow trout's sensitivity to the lytic bacterial challenge of Salmonicida and in the population prevented with the lysozyme dimer composition indicate that the drug induces short-term protection against these microbial challenges. In addition, protection was higher in groups receiving KLP-602 two or three times than in groups treated once.

conclusion.

Application of high-purity lysozyme dimers in mollusks led to significant protection and general defense against the microbial challenge of Salmonella. This observation is particularly important as a prophylactic treatment for fish farming in order to minimize the loss of fish due to frequent fish diseases, especially in spring.

Example 4

Therapeutic application of lysozyme dimers has induced general stimulation of the immune system of fish.

Testing process

The 200 rainbow clusters are used for comparative studies of general defense mechanisms and the application of dimer lysozyme (KLP-602) during the naturally occurring infection of IPNV (Infectious Pancreatic Lisa Virus). After identification of IPNV infection, a portion of the fish was treated with KLP-602 and the control was left untreated. The main variables characterized by general cellular and humoral defense mechanisms were analyzed by immunological and hematological methods. These methods include total leukocyte count, relative leukocyte count, phagocytosis of PMN and NM cells, respiratory rupture activity and potential lethality, MPO activity in neutrophils, lysozyme and ceruloplasmin activity in plasma and total protein during 2 months of observation. Gamma-globulin levels are determined weekly.

Among them, the rainbow trout population fed a diet containing general immunostimulatory KLP-602 in an amount of 20 μg / kg body weight. The lyophilized KLP602 and the nutrients are applied as a solid (pellets) or dissolved in fish pond water in an aqueous solution for 7 days. The drug can be absorbed through fish gills.

The weekly intervals are then examined for changes in normal cell and fluid defense mechanisms. At each time, 10 fish are randomly selected for immunological testing to confirm IPNV. In addition, all fish are monitored daily for abnormal behavior, morphological changes and mortality.

result.

Immunosuppressive effects were observed in fish naturally infected with IPNV. During the overall observation time, the parameters indicating normal cell and humoral defense mechanisms decreased significantly, while some variables, such as celluloplasmin levels, respiratory rupture activity, immunoslobulin, compared to controls after 1 week of lysozyme dimer delivery Variables, including levels and lysozyme activity, increased significantly (eg, ceruloplasmin levels, 31% increase; respiratory burst activity 33% increase). After the application of KLP-602, an immunostimulatory effect was observed on the general defense mechanism. In addition, the mortality rate was low in the population receiving KLP-602.

conclusion.

Fish treated with food containing lysozyme dimer (KLP-602) showed beneficial changes in general immunological defense mechanisms. As a reduced lethal example, it is associated with indicating protection against spontaneous infection by IPNV, which is strongly recommended for developing and optimizing the prevention and treatment of fish diseases by administration of the lysozyme dimers mentioned herein.

Claims (15)

In pharmaceutical compositions comprising lysozyme dimers, which are used to prevent or treat leukemia by promoting non-specific cell or humoral response mechanisms in the immune system of humans or animals, wherein the facilitation increases phagocytic activity, A pharmaceutical composition for treating leukemia, wherein the composition is increased by increasing at least one variable selected from among roplasmin levels, respiratory releasing activity, immunoglobulin levels, and lysozyme, or the facilitating response regulates free oxygen radical levels in serum. Pharmaceutical compositions comprising lysozyme dimers, which are used to prevent or treat hair growth-related diseases by promoting non-specific cell or humoral response mechanisms in the immune system of humans or animals, wherein the facilitation reaction increases phagocytic activity. Increase in at least one variable selected from ceruloplasmin level, respiratory release activity, immunoglobulin level, lysozyme, or the facilitation reaction is characterized by regulating free oxygen radical levels in serum. Therapeutic pharmaceutical composition. In pharmaceutical compositions comprising lysozyme dimers as active ingredients used to prevent or treat fish diseases such as boil species and IPNV infection by promoting non-specific cell or humoral response mechanisms in fish, wherein the facilitating response is a phagocytic activity. Boil species characterized by increasing, at least one variable selected from ceruloplasmin levels, respiratory releasing activity, immunoglobulin levels, lysozyme, or promoting reactions to control free oxygen radical levels in serum; Pharmaceutical composition for the treatment of fish diseases such as IPNV infection. The lysozyme dimer according to any one of claims 1, 2 and 3, wherein the lysozyme dimer contains less than 10% (by weight) of unwanted by-products, and the monomeric lysozyme is not present. Pharmaceutic composition. The pharmaceutical composition according to claim 1, wherein the disease is a virus-induced leukemia. 6. The pharmaceutical composition according to claim 5, wherein the pathogenic virus is a retrovirus. The pharmaceutical composition according to claim 2, wherein the hair growth-related disease is alopecia areata. 4. The pharmaceutical composition according to claim 3, wherein the disease occurs in salmon and fish. The pharmaceutical composition according to any one of claims 1, 2 and 3, which is administered to a subject to be treated in a single or repeated dose of 0.005 to 0.5 mg / kg body weight. The pharmaceutical composition according to any one of claims 1, 2 and 3, which is administered to a subject to be treated in a single or repeated dose of 0.01 to 0.1 mg / kg per body weight. The pharmaceutical composition according to any one of claims 1, 2 and 3, wherein the pharmaceutical composition comprising the lysozyme dimer is prepared in the form of a gel, ointment or liquid composition for topical, oral or extranasal administration, wherein the pharmaceutical composition is physiologically A pharmaceutical composition further comprising at least one of an acceptable solvent, a carrier and a suitable preservative. The pharmaceutical composition according to claim 11, wherein the active substance lysozyme dimer contains 0.01 to 10 mg / ml. 12. The pharmaceutical composition according to claim 11, wherein the active substance lysozyme dimer contains 0.01 to 1 mg / ml. The pharmaceutical composition according to any one of claims 1, 2 and 3, wherein the pharmaceutical composition is prepared in a solid suitable for oral administration and further contains at least one of a carrier and a preservative. 15. The pharmaceutical composition according to claim 14, wherein the active lysozyme dimer is contained in an amount of 0.01 to 10 mg / kg.

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